An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5
A duplex real-time quantitative reverse transcription-polymerase chain reaction (dqRT-PCR) assay was successfully developed to simultaneously detect canine parainfluenza virus 5 (CPIV5) and a canine endogenous internal positive control (EIPC) in canine clinical samples. Two sets of primers and probe...
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| Format: | Article |
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MDPI AG
2023-02-01
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| Series: | Veterinary Sciences |
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| Online Access: | https://www.mdpi.com/2306-7381/10/2/142 |
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| author | Gyu-Tae Jeon Hye-Ryung Kim Yeun-Kyung Shin Oh-Kyu Kwon Hae-Eun Kang Oh-Deog Kwon Choi-Kyu Park |
| author_facet | Gyu-Tae Jeon Hye-Ryung Kim Yeun-Kyung Shin Oh-Kyu Kwon Hae-Eun Kang Oh-Deog Kwon Choi-Kyu Park |
| author_sort | Gyu-Tae Jeon |
| collection | DOAJ |
| description | A duplex real-time quantitative reverse transcription-polymerase chain reaction (dqRT-PCR) assay was successfully developed to simultaneously detect canine parainfluenza virus 5 (CPIV5) and a canine endogenous internal positive control (EIPC) in canine clinical samples. Two sets of primers and probes for the CPIV5 L and canine 16S rRNA genes were included in the dqRT-PCR assay to detect CPIV and monitor invalid results throughout the qRT-PCR process. The developed dqRT-PCR assay specifically detected CPIV5 but no other canine pathogens. Furthermore, <i>16S rRNA</i> was stably amplified by dqRT-PCR assay in all samples containing canine cellular materials. The assay’s sensitivity was determined as below ten RNA copies per reaction, with CPIV5 L gene standard RNA and 1 TCID<sub>50</sub>/mL with the CPIV5 D008 vaccine strain, which was 10-fold higher than that of the previous HN gene-specific qRT-PCR (<i>HN</i>-qRT-PCR) assays and was equivalent to that of the previous N gene-specific qRT-PCR (<i>N</i>-qRT-PCR) assays, respectively. Moreover, the Ct values of the CPIV5-positive samples obtained using the dqRT-PCR assay were lower than those obtained using the previous <i>HN</i>- and <i>N</i>-qRT-PCR assays, indicating that the diagnostic performance of the dqRT-PCR assay was superior to those of previous <i>HN</i>- and <i>N</i>-qRT-PCR assays. The calculated Cohen’s kappa coefficient values (95% confidence interval) between dqRT-PCR and the <i>HN-</i> or <i>N</i>-specific qRT-PCR assays were 0.97 (0.90–1.03) or 1.00 (1.00–1.00), respectively. In conclusion, the newly developed dqRT-PCR assay with high sensitivity, specificity, and reliability will be a promising diagnostic tool for the detection of CPIV5 in clinical samples and useful for etiological and epidemiological studies of CPIV5 infection in dogs. |
| first_indexed | 2024-03-11T08:02:14Z |
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| institution | Directory Open Access Journal |
| issn | 2306-7381 |
| language | English |
| last_indexed | 2024-03-11T08:02:14Z |
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| spelling | doaj.art-d2f51d782c9d4390a778793e0d0b4aaa2023-11-16T23:46:11ZengMDPI AGVeterinary Sciences2306-73812023-02-0110214210.3390/vetsci10020142An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5Gyu-Tae Jeon0Hye-Ryung Kim1Yeun-Kyung Shin2Oh-Kyu Kwon3Hae-Eun Kang4Oh-Deog Kwon5Choi-Kyu Park6Animal Disease Intervention Center, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Republic of KoreaAnimal Disease Intervention Center, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Republic of KoreaForeign Animal Disease Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Republic of KoreaForeign Animal Disease Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Republic of KoreaForeign Animal Disease Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Republic of KoreaAnimal Disease Intervention Center, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Republic of KoreaAnimal Disease Intervention Center, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Republic of KoreaA duplex real-time quantitative reverse transcription-polymerase chain reaction (dqRT-PCR) assay was successfully developed to simultaneously detect canine parainfluenza virus 5 (CPIV5) and a canine endogenous internal positive control (EIPC) in canine clinical samples. Two sets of primers and probes for the CPIV5 L and canine 16S rRNA genes were included in the dqRT-PCR assay to detect CPIV and monitor invalid results throughout the qRT-PCR process. The developed dqRT-PCR assay specifically detected CPIV5 but no other canine pathogens. Furthermore, <i>16S rRNA</i> was stably amplified by dqRT-PCR assay in all samples containing canine cellular materials. The assay’s sensitivity was determined as below ten RNA copies per reaction, with CPIV5 L gene standard RNA and 1 TCID<sub>50</sub>/mL with the CPIV5 D008 vaccine strain, which was 10-fold higher than that of the previous HN gene-specific qRT-PCR (<i>HN</i>-qRT-PCR) assays and was equivalent to that of the previous N gene-specific qRT-PCR (<i>N</i>-qRT-PCR) assays, respectively. Moreover, the Ct values of the CPIV5-positive samples obtained using the dqRT-PCR assay were lower than those obtained using the previous <i>HN</i>- and <i>N</i>-qRT-PCR assays, indicating that the diagnostic performance of the dqRT-PCR assay was superior to those of previous <i>HN</i>- and <i>N</i>-qRT-PCR assays. The calculated Cohen’s kappa coefficient values (95% confidence interval) between dqRT-PCR and the <i>HN-</i> or <i>N</i>-specific qRT-PCR assays were 0.97 (0.90–1.03) or 1.00 (1.00–1.00), respectively. In conclusion, the newly developed dqRT-PCR assay with high sensitivity, specificity, and reliability will be a promising diagnostic tool for the detection of CPIV5 in clinical samples and useful for etiological and epidemiological studies of CPIV5 infection in dogs.https://www.mdpi.com/2306-7381/10/2/142canine parainfluenza virus 5duplex real-time quantitative RT-PCRL geneendogenous internal positive controlCIRD |
| spellingShingle | Gyu-Tae Jeon Hye-Ryung Kim Yeun-Kyung Shin Oh-Kyu Kwon Hae-Eun Kang Oh-Deog Kwon Choi-Kyu Park An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5 Veterinary Sciences canine parainfluenza virus 5 duplex real-time quantitative RT-PCR L gene endogenous internal positive control CIRD |
| title | An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5 |
| title_full | An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5 |
| title_fullStr | An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5 |
| title_full_unstemmed | An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5 |
| title_short | An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5 |
| title_sort | improved duplex real time quantitative rt pcr assay with a canine endogenous internal positive control for more sensitive and reliable detection of canine parainfluenza virus 5 |
| topic | canine parainfluenza virus 5 duplex real-time quantitative RT-PCR L gene endogenous internal positive control CIRD |
| url | https://www.mdpi.com/2306-7381/10/2/142 |
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